Humanized anti-IL-10 antibodies for the treatment of systemic lupus erythematosus (SLE)

ABSTRACT

Provided is a humanized or chimeric antibody or fragment thereof capable of binding to interleukin-10 (Th-10), wherein said antibody or fragment thereof is capable of being administered to a subject in the absence of an intolerable increase in the level of pro-inflammatory cytokines. Further provided are methods of treatment involving the use of the antibody or fragment thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a national stage application of International Patent ApplicationPCT/EP2010/068562, which claims priority to GB 0920933.9, filed Nov. 30,2009, GB 0920940.4, filed Nov. 30, 2009, and GB 0920942.0, filed Nov.30, 2009, the disclosures of each of which are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention is concerned with interleukin-10 (IL-10) and IL-10specific agents. In particular, the present invention involves humanizedIL-10 antibodies and their uses. The invention further envisages amethod of treatment of systemic lupus erythematosus (SLE).

BACKGROUND TO THE INVENTION

Systemic lupus erythematosus (SLE) is regarded as an autoimmune disease,in which abnormal hyperactivity of B lymphocytes and massive abnormalproduction of immunoglobulin gamma (IgG) auto-antibodies plays a keyrole. This pathological process results in sequestration and destructionof Ig-coated cells, fixation and cleaving of complement proteins, andrelease of chemotaxins, vasoactive peptides and destructive enzymes intotissues (Hahn B H. Systemic Lupus Erythematosus. In: Kasper D L,Braunwald E, Fauci A S, Hauser S L, Longo D L, Jameson, J L, editors.In: Harrison's Principles of Internal Medicine (16th edition). New York(US): McGraw-Hill; 2005. pp. 1960-1967).

SLE is characterized by diverse manifestations. In the course of thedisease a total of 95% of patients complain of musculoskeletal disease,80% show cutaneous lesions, 85% haematological disease, 60% neurologicaldisorders, 60% cardiopulmonary disease, 30% to 50% renal disease, 40%gastrointestinal disease, 15% thrombosis and 15% ocular disease. Thevast majority of the patients (95%) also suffer from systemic symptomssuch as fatigue, malaise, fever, anorexia, and weight loss, which arepresent most of the time. Most patients experience disease periods offlares alternating with remissions. Permanent remissions (absence ofsymptoms with no treatment) are very rare. More than 50 years ago mostpatients diagnosed with SLE lived less than 5 years. Nowadays, 10 yearsurvival is over 90%, mainly based on earlier diagnosis, symptomaticanti-inflammatory and immune-suppressive treatment. The common cause ofdeath is infection as a result of immune-suppression (Hahn 2005).

Antimalarial, anti-inflammatory and immunosuppressive drugs haveroutinely been used in the treatment of SLE. Non-steroidalanti-inflammatories have been supplemented with corticosteroids when thesymptoms become difficult to control. Further, active SLE, with majororgan involvement, requires aggressive therapy with cyclophosphamide.

Up to now, there is no causative treatment available to cure SLE and/orimprove patients' quality of life on a long term basis. However, recentadvances in antibody technology and the further identification offactors underlying this autoimmune disease have opened up thepossibility of using monoclonal antibodies as a treatment option. Inparticular, a favourable approach to treat SLE would be a specifictreatment interacting or correcting the pathological immune responseresulting in the massive overproduction of polyclonal auto-antibodies.Since the pathogenesis of SLE primarily involves dysregulated B cells,monoclonal antibodies capable of targeting B-cells are of specialinterest. As noted by Robak and Robak (Current Drug Targets, 2009, No.10, pages 26-37) potential B-cell surface antigen targets are CD19,CD20, CD21 and CD22. Further, IL-10, IL-1ra, IL-12 (Capper et al., Clin.Exp. Immunol. 2004 November; 138(2):348-56), and IL-6 (Chun et al., J.Clin. Immunol. 2007 September; 27(5):461-6) are important cytokines inregulating immune response and are especially raised during flares inSLE patients. Plasma levels of IL-10 and auto-antibodies againstdouble-stranded DNA (dsDNA) often mirror disease activity in patientswith SLE. Raised IL-10 levels correlated with disease activity in SLEpatients (Park et al., Clin. Exp. Rheumatol. 1998 May-June;16(3):283-8). However, IL-10 is a cytokine with pleiotropic effects onthe immune system and is also known to be involved in reducingproinflammatory responses.

Clinical trials with monoclonal antibodies have been conducted in SLEpatients. In particular, several trials have involved the antibodyRituximab, a chimeric mouse anti-CD20 monoclonal antibody used for thetreatment of non-Hodgkin's lymphoma. As noted by Robak and Robak (2009),the results of these trials show high activity of this antibody in SLEpatients, and several new antibodies targeting CD20 have been developed;Ofatumumab, IMMU-106 and GA-101. Further clinical trials reportingactivity of monoclonal antibodies in SLE have been completed with theanti-CD22 antibody, Epratuzumab, the anti-TNFα antibody, Infliximab, theanti-IL-10 antibody, B-N10 (Llorente et al., Arthritis Rheum. 2000August; 43(8): 1790-800), the anti-CD40L antibodies, IDEC 131 and BG9588, the BLYS inhibitor, Belimumab, the anti-IL6 receptor antibody,Toclimumab, and the anti-C5 antibody Eculizumab.

It is the aim of the present invention to provide further agents, and inparticular antibodies, having utility in this area.

Accordingly the present invention provides a humanized or chimericantibody or fragment thereof capable of binding to interleukin-10(IL-10), wherein said antibody or fragment thereof is capable of beingadministered to a subject in the absence of an intolerable increase inthe level of pro-inflammatory cytokines.

Since IL-10 represents an anti-inflammatory cytokine, one would expectupon blocking a dramatic increase of cytokines. For the murine IL-10antibody, B-N10, where one can observe in unstimulated cell cultures(which reflects the in vivo situations in healthy individuals) anincrease in proinflammatory cytokines such as IL-6 or TNF alpha.However, the inventors have surprisingly found that when the antibody ofthe present invention is applied to the cells in vitro, and administerdin vivo, cytokine release is much lower. This lower cytokine release isadvantageous as, as a result, the antibody of the present invention ismore tolerable to individuals to which it is administered.

The invention will be illustrated by way of example only, with referenceto the following Figures, in which:

FIG. 1A shows the amino acid sequence of the light chain variable regionof the murine B-N10 antibody (SEQ ID No:2). The hypervariablecomplementarity-determining regions (CDRs) are underlined (wherein LCDR1is SEQ ID No: 4; LCDR2 is SEQ ID No: 5; and LCDR3 is SEQ ID No:6).

FIG. 1B shows the amino acid sequence of the heavy chain variable regionof the murine B-N10 antibody (SEQ ID No: 3). The hypervariablecomplementarity-determining regions (CDRs) are underlined (wherein HCDR1is SEQ ID No: 7; HCDR2 is SEQ ID No: 8; and HCDR3 is SEQ ID No:9).

FIG. 2A shows the nucleotide sequence encoding the light chain variableregion of the murine B-N10 antibody (SEQ ID No: 10).

FIG. 2B shows the nucleotide sequence encoding the heavy chain variableregion of the murine B-N10 antibody (SEQ ID No: 11).

FIG. 3 shows the amino acid sequence of the murine B-N10 light and heavychain variable regions (SEQ ID Nos: 12 and 13, respectively) togetherwith the sequences taken from A17 (SEQ ID No: 14), JK1 (SEQ ID No: 15),3-66+04 (SEQ ID No: 16) and JH4 (SEQ ID No: 17) and the variable regionshVL1 to hVL12 (SEQ ID Nos: 18 to 29) and the variable regions hVH1 tohVH29 (SEQ ID Nos: 30 to 58) generated during the humanization of themurine B-N10 antibody.

FIG. 4 provides a comparison of the antigen binding properties of thehumanized antibody variants in comparison to a chimeric cB-N10 antibodyusing the hIL-10 antigen ELISA.

FIG. 5 provides the result of the determination of the bindingproperties of the three humanized variants, hVH20/hVL7, hVH20/hVL8 andhVH26/hVL7, in comparison to the chimeric B-N10 antibody using purifiedantibody preparations.

FIG. 6A shows the level of TNFalpha release from whole blood culturesfrom healthy volunteers after incubation with B-N10 compared to BT-063(at 50 μg/ml).

FIG. 6B shows the level of TNFalpha release from whole blood culturesfrom SLE patients after incubation with B-N10 compared to BT-063 (at 50μg/ml).

FIG. 7A shows the level of IL-6 release from whole blood cultures fromhealthy volunteers after incubation with B-N10 compared to BT-063 (at 50μg/ml).

FIG. 7B shows the level of IL-6 release from whole blood cultures fromSLE patients after incubation with B-N10 compared to BT-063 (at 50μg/ml).

FIG. 8A shows the level of IL-1beta release from whole blood culturesfrom healthy volunteers after incubation with B-N10 compared to BT-063(at 50 μg/ml).

FIG. 8B shows the level of IL-1beta release from whole blood culturesfrom SLE patients after incubation with B-N10 compared to BT-063 (at 50μg/ml).

FIG. 9A shows the level of IFN gamma release from whole blood culturesfrom healthy volunteers after incubation with B-N10 compared to BT-063(at 50 μg/ml).

FIG. 9B shows the level of IFN gamma release from whole blood culturesfrom SLE patients after incubation with B-N10 compared to BT-063 (at 50μg/ml).

FIG. 10 shows the overall structure of the Fab fragment of BT-063binding IL-10. IL-10 and the Fab fragment are shown as a ribbonrepresentation.

FIG. 11 shows the Fab fragment of BT-063 addresses the same binding siteon IL-10 as the IL-10 receptor. IL-10, IL-10R1 and the Fab fragment areshown as a ribbon representation.

FIG. 12 shows a graph of mean cmax of cytokine concentration in plasmaversus dosage after administration of BT-063 to healthy volunteers.

FIG. 13 shows a graph of mean IL-10 concentration in plasma over timeafter in vivo administration of BT-063 in healthy volunteers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a humanized or chimeric antibody orfragment thereof capable of binding to interleukin-10 (IL-10), and theuse of this antibody or fragment thereof in the treatment of medicalconditions that are mediated by an elevated level or activity of IL-10.

Human IL-10 is a homodimer with a molecular mass of 37 kDa. Each monomerconsists of 160 amino acids and has a molecular mass of 18.5 kDa. TheIL-10 dimer interacts with the IL-10R receptor alpha (IL-Rα or IL-10R1)and subsequently recruits IL-10 receptor beta (IL-10Rβ or IL-10R2) intothe complex. The receptor is expressed on a variety of cells, inparticular immune cells (Asadullah et al., Pharmacol. Rev. 2003 June;55(2):241-69) including most hematopoietic cells such as monocytes,macrophages, and T- and B-lymphocytes, but is also expressed onnon-hemopoietic cells, such as epidermal cells or keratiocytes. Thebinding of IL-10 receptor alpha by IL-10 and the recruitment of IL-10receptor beta leads to signal transduction via Jak1 and Tyk2 tyrosinekinases and subsequently to activation of transcription factors of theSTAT family. Various cellular sources of IL-10 are known, such as Thelper cells, regulatory T cells, monocytes, macrophages, B cells,eosinophils, mast cells, keratinocytes, dendritic cells and even cancercells. IL-10 functions on B cells range from prevention of apoptosis,enhancement of proliferation, class switching events and differentiationinto plasma cells (Asadullah et al., Pharmacol. Rev. 2003 June;55(2):241-69).

The present invention provides a humanized or chimeric antibody orfragment thereof capable of binding to interleukin-10 (IL-10), whereinsaid antibody or fragment thereof is capable of being administered to asubject in the absence of an intolerable increase in the level ofpro-inflammatory cytokines.

In particular, it is known that the administration of therapeuticantibodies can lead to an intolerable increase in the levels ofpro-inflammatory cytokines which lead to adverse side effects in thesubject. In particular, the intolerable increase can cause the reddeningof the skin and induce a fever or flu-like symptoms, including anincrease in body temperature. Accordingly, in a preferred aspect of theinvention the antibody or antibody fragment is one which is capable ofbeing administered to a subject in the absence of an increase in bodytemperature of greater than 2° C.

Further, the antibody or fragment thereof does not cause a substantialincrease in the amount of pro-inflammatory cytokines in the subject'sblood plasma after administration. Levels of these cytokines which areintolerable are usually several times greater than the cytokines upperlimit of normal (ULN), where the ULN is defined as the mean level of acytokine(s) measured in a subject cohort plus 2× standard deviations.

Accordingly, in a preferred aspect of the invention the antibody orfragment thereof is capable of being administered to a subject in theabsence of an increase of the pro-inflammatory cytokine which is greaterthan 500%, more preferably greater than 300%, of the upper limit ofnormal (ULN). In other words, the antibody or fragment thereof causes anincrease in the level of a pro-inflammatory cytokine which is less than500%, more preferably less than 300% of the ULN for that cytokine. It ispreferred that the pro-inflammatory cytokine is at least one, andpreferably all, of TNF-α, IFN-γ, and IL-1 beta. Alternatively, thepro-inflammatory cytokine and is not IL-6 or IL-8.

Still further, it is particularly preferred that the antibody orfragment thereof of the present invention is capable of inducing IL-1receptor antagonist (IL-1ra) in the subject, which leads to ananti-inflammatory response.

In a preferred aspect of the present invention the humanized antibody orfragment thereof does not elicit a greater than 500% increase in thelevel of a pro-inflammatory cytokines, especially TNFalpha, IL-1beta,IL-6 released from a PBMC, more particularly an immune cell, whencontacted with said cell in vitro.

The present inventors have surprisingly found that the humanizedantibody or fragment thereof of the invention elicits a smaller increasein the level of pro-inflammatory cytokines in in vitro assays than themurine B-N10 antibody.

The level of pro-inflammatory cytokine release can be determined by invitro studies utilizing human whole blood cultures, such as thosestudies described below in Example 5, or isolated immune cells. Inparticular, the methods comprise the steps of: (a) incubating a cellculture with the antibody or fragment thereof; (b) and determining thelevel of at least one pro-inflammatory cytokine.

The human whole blood cultures can be taken from healthy or diseasedpatients, such as those suffering from SLE. The peripheral bloodmononuclear cell (PBMC) can be an immune cell, and, in particular,selected from macrophages, monocytes, dendritic cells, T helper cellsand B cells.

The at least one proinflammatory cytokine can be selected from,interleukin-1 beta (IL-1beta), IL-1 alpha, IL-6, or tumor necrosisfactor alpha (TNF-alpha) or T helper cytokines (interferon gamma,IFN-gamma, IL-4), macrophage cytokine (IL-12), chemokines (IL-8, MCP-1)respectively. The levels of such cytokines which are released can bemeasured in the cell culture supernatant using methods which aregenerally known in the art.

More particularly this in vitro method can comprise the steps of:

-   -   a) contacting 50 μg/ml of the antibody or fragment with a human        whole blood culture;    -   b) incubating said antibody or fragment with said whole blood        culture at 37° C. for 48 hours;    -   c) determining the quantity of one or more pro-inflammatory        cytokines in the culture.

In particular, the method is run simultaneously with human whole bloodculture which has not been contacted with the antibody Preferably, whencompared to this control the antibody or antibody fragment of thepresent invention causes a less than 500% increase in the level of thecytokine, more preferably a less than 300% increase in the level of thecytokine. It is preferred that the cytokine in this method is not IL-6or IL-8.

Cytokines can be detected in the culture with a Multiplex BeadImmunoassays (solid-phase protein assays which are carried out in96-well filter plates). The method relies on the use of beads which arelinked, firstly, with a specific antibody against a specific cytokineand, secondly, exhibit defined spectral characteristics. This enablesthe beads to be identified specifically and thus to assign them to theknown, linked antibody. The cytokine bound to the bead can be quantifiedby means of further binding of the cytokines by a detection antibody.This makes it possible to detect simultaneously up to 10 humancytokines, including GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10,IFN-γ and TNF-α. Filter plates are used for this purpose into whosemoistened wells the samples, standards and the bead solution arepipetted. After incubation, the liquid is aspirated off with a vacuumpump, the beads left behind are washed, and a mixture of biotinylateddetection antibodies is added. Unbound detection antibodies are removedby aspirating off the liquid and washing the beads remaining in thewell, and then streptavidin-conjugated R-phycoerythrin(streptavidin-RPE) is added. This recognises the biotinylated detectionantibodies during incubation, and after washing again, the immunecomplexes formed can be analysed by the Bio-Plex 200 System. Theimmunoassay is analysed using the Bio-Plex 200 device. This uses twolaser systems, which, firstly, identify the beads on the basis of theirspectral characteristics and, secondly, determine the quantity of boundcytokines by detection using a secondary antibody. The quantification ofthe bound quantity is achieved by means of standards run in parallelwith known quantities of cytokines.

The present invention also provides a humanized or chimeric antibody orfragment thereof capable of binding to interleukin-10 (IL-10), whereinsaid antibody or fragment thereof is capable of increasing plasma levelsof IL-10 when administered to a subject.

As demonstrated in Example 8 below, while the antibody of the presentinvention can be admininstered in the absence of an intolerable increasein the level of pro-inflammatory cytokines, administration increases theamount of IL-10 detectable in plasma samples in a dose dependentfashion. This finding, that IL-10 levels would increase when aneutralizing antibody is applied, is unexpected. It would be usual toexpect that the administration of a cytokine neutralizer would reducethe level of free cytokine (Strand et al., Nature Reviews DrugDiscovery, 2007, Vol. 6, pages 75-92). While not wishing to be bound bytheory it is thought that binding of IL-10 by the antibody preventsIL-10 binding to the IL-10 receptor and triggers a negative feedbackloop which causes B-cells to produce more IL-10. Nevertheless, theupregulation does not prevent the therapeutic utility of the antibody ofthe present invention since, also as demonstrated in Example 8, theantibody is safe to administer at levels high enough to neutralize allIL-10.

Within the present application the term “chimeric antibody” refers toantibodies in which a portion of the heavy and/or light chain isidentical with sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the antibody is identical with sequences in antibodiesderived from a different species, antibody class or subclass. It isparticularly preferred that the CDRs of a chimeric antibody have oneorigin, while the remainder of the antibody has a different origin. Inparticular, in the present invention the chimeric antibody may be ahumanized antibody in which the antigen binding sequences/variabledomains of a non-human antibody have been grafted onto human antibodyframework regions.

Within the present application the term “fragment” refers to a fragmentor a derivative of an antibody that still retains the desired biologicalactivity. The fragment will generally comprise the antigen bindingregion of the antibody and, in particular, the Fab, Fab′, F(ab)′₂, Fvand scFv fragments, as well as multivalent antibody derivatives, inparticular diabodies or tandem diabodies. The fragment preferably is atleast 25, more preferably 50 and still more preferably 200 to 500 aminoacids. Alternatively the fragments can be defined as those having ofsize of between 30 KDa and 150 kDa. Further, the antibody fragments mayinvolve two or more peptide/polypeptide chains. For example an Fabfragment comprising two chains of between 200 and 300 amino acids inlength each or TandAbs® (tetravalent bispecific antibody formats)comprising two chains of between 400 and 500 amino acids in length each.

It is a preferred feature of the invention that the antibody or fragmentthereof is derived from the murine B-N10 antibody or the humanizedBT-063 (variant hVH26/hVL7) antibody described herein. In particular,such an antibody or fragment thereof will comprise CDRs being at least80% identical to those of CDR1, CDR2 and CDR3 of B-N10/BT-063 variablelight chain and/or comprises amino acid sequences at least 80% identicalto those of CDR1, CDR2 and CDR3 of B-N10/BT-063 variable heavy chain.The amino acid sequence of the murine CDRs is shown in FIG. 1. The aminoacid sequence of the BT-063 CDRs is shown in Example 6. More preferablythe sequences will be at least 90%, or at least 95% identical to thoseof the CDRs of the B-N10/BT-063 antibody. The X ray crystallographystudies described in Example 6 herein indicate which residues within theCDRs are important for binding to IL-10.

Alternatively, the antibody or fragment of the invention, while stillbeing derived from the B-N10/BT-063 antibody, can comprise an amino acidsequence of CDR1, CDR2 and CDR3 of the B-N10/BT-063 variable light chainand/or an amino acid sequence of CDR1, CDR2 and CDR3 of B-N10/BT-063variable heavy chain, optionally with variation in these sequences whichdoes not substantially alter the affinity and/or specificity of theantibody or fragment thereof. In particular, the variations in thesequence do not reduce the affinity or specificity of the antibody orfragment for IL-10 as compared to that of an antibody or fragmentcomprising the CDRs of the murine B-N10 antibody or the BT-063 (varianthVH26/hVL7) antibody.

In a specific embodiment the present invention provides a humanized orchimeric antibody or fragment thereof which comprises the amino acidsequences of CDR1, CDR2 and CDR3 of the B-N10/BT-063 variable lightand/or heavy chains. More preferably the present invention provides ahumanized or chimeric antibody or fragment thereof which comprises theamino acid sequences of the variable domains of the murine antibodyB-N10, as shown in FIG. 1. Most preferably the antibody or fragmentcomprises one or both of the amino acid sequences of the variabledomains of BT-063 (SEQ ID No: 69 and SEQ ID No: 70).

Generally, the antibody of the invention further comprises a humanconstant region (Fc). This can be selected among constant domains fromany class of immunoglobulines, including IgM, IgG, IgD, IgA and IgE, andany isotype, including IgG1, IgG2, IgG3 and IgG4. Preferred constantregions are selected among constant domains of IgG, in particular, IgG1.

Other Products

The present invention further provides nucleic acid sequences encodingthe antibody or antibody fragments described above. The nucleic acidsequences may be DNA or RNA but are preferably DNA. The sequences may beused within expression cassettes or vectors, and are of particular usein producing the antibodies and fragments thereof disclosed herein.

The invention further provides host cells transformed with thesepolynucleotides, expression cassettes or vectors. Suitable host cellscan be both prokaryotic and eukaryotic.

Alternatively, the host cell can be a hybridoma obtained by fusing acell producing an antibody of the present invention with a myeloma cell.

The host cells described above can be utilized in a method for theproduction of an antibody or fragment thereof. In particular, such amethod may comprise a step of culturing the host cell in a suitableculture medium under conditions allowing the expression of the antibodyor fragment thereof and separating the antibody or fragment from theculture medium. Methods of this type are well known and described in theart.

Medical Uses

The antibodies and fragments thereof described herein have utility inthe treatment of diseases or medical conditions which are mediated by anelevated level or activity of IL-10. As a result, provided is a methodfor treating or preventing a medical condition in a subject, wherein themedical condition is mediated by an elevated level or activity of IL-10,comprising administering a therapeutically effective amount of theantibody or fragment thereof described herein.

In particular, the medical condition which is mediated by an elevatedlevel or activity of IL-10 is SLE. Accordingly, the present inventionalso provides an antibody or fragment thereof as described herein foruse in the treatment of SLE.

Further examples are thrombocytopenic purpura, lupus nephritis, HIV,hCMV and hepatitis C. The treatment of tumor cells depending on IL-10 bydirect support of proliferation or suppression of immune response isanother example.

A further embodiment of the invention is a pharmaceutical compositioncomprising the antibody or fragment thereof described above with apharmaceutically acceptable carrier or diluent. In one embodiment thecomposition comprises liposomes to which the antibody or fragmentthereof is coupled.

Such compositions can be administered to the patient parenterally,intravenously or subcutaneously. Preferably in the treatment of SLE theantibody or fragment thereof is administered intravenously orsubcutaneously.

The antibodies and fragments thereof are of particular use in thetreatment of disease, since, as they do not cause a significant increasein the levels of pro-inflammatory cytokines, they do not require theco-administration of a corticosteroid to prevent a “cytokine storm”which would be intolerable to the patient.

Thus, in a particular aspect the present invention provides a method fortreating or preventing a medical condition in a subject, wherein themedical condition is mediated by an elevated level or activity of IL-10,comprising administering a therapeutically effective amount of anantibody or fragment thereof as described herein to said subject,wherein the subject is not simultaneously or separately treated with acorticosteroid, i.e. the patient is one who is not concurrently beingtreated with a corticosteroid. In an embodiment of this preferred aspectthe medical condition mediated by an elevated level or activity of IL-10is SLE.

Corticosteroids are currently used to treat SLE in many patients.However, this aspect of the invention has particular utility in thetreatment of patients where the administration of corticosteroids is nolonger desirable.

Non-Medical Uses

Still further provided is a labeled humanized or chimeric antibody orfragment thereof comprising the antibody or fragment thereof describedherein and a label. The label may be of any suitable type known in theart for detecting the presence of an antibody in a sample. Inparticular, the label may be a fluorescent label.

The labeled or unlabeled antibody, and in particular the labeledantibody, has specific utility in an in vitro method for detecting thepresence of IL-10 in a sample. The method may comprise a step ofcontacting the unlabeled or labeled antibody or fragment thereof withthe sample, washing the sample to remove antibody and fragments thereofwhich are not bound to the sample (i.e. unbound antibody or antibodyfragments) and detecting the presence of the antibody (or fragment), forexample via the label, in the sample.

Alternatively, the unlabeled antibody or fragment may be used for an invitro method for neutralizing IL-10 in a sample. Such a method comprisesthe steps of contacting the sample with the antibody or fragment thereofso as to bind the antibody or fragment thereof to the IL-10.

The invention will now be described further in relation to the followingspecific embodiments.

EXAMPLES Example 1 Characterisation of Murine Anti-IL10 Antibody B-N10

1.1. Isolation of DNA Encoding the Variable Antibody Domains of B-N10

For the identification of the variable sequences of murine BN-10 cellpellets were used. The samples (3×B-N10, Passage 3, 1×10⁷ cells) werestored at −80° C. until mRNA was isolated from the cells and, after cDNAsynthesis, the variable sequences of B-N10 were amplified by PCR andthen cloned.

In total 14 clones were sequenced (SEQ Laboratories, Gottingen) andanalysed for both the variable light chain and for the variable heavychain. The variable sequences of the B-N10 were determinedunequivocally. Deviations occurred only in the N-terminal primer regions(see Table 1). In the case of the variable heavy chain, the sequencevariant QVQLKQ (SEQ ID No: 59) occurred nine times in the primer region,but other variations occurred only once or twice. This variant waschosen for subcloning. In the case of the variable light chain, twovariants were present in equal proportions. After comparing thesequences with the murine germ line sequences, the cr1 sequence withonly 3 mutations exhibited great homology with the identified VLsequence. This means that the DVLMTQ (SEQ ID No: 60) sequence is mostprobably the correct sequence. The sequence DIVMTQ (SEQ ID No: 61) istypical of another class of germ line sequence and was thereforeexcluded.

TABLE 1 Occurrence of N-terminal sequencevariants of the sequenced variable lightand heavy chain of B-N10. The chosensequences are indicated in bold type. Sequence Number VariableDIVMTQ (SEQ ID No: 61) 5 light chain DVLMTQ (SEQ ID No: 60) 5DVLMTR (SEQ ID No: 62) 1 DIVITQ (SEQ ID No: 63) 1 DIVLTQ (SEQ ID No: 64)2 Variable QVQLKQ (SEQ ID No: 59) 9 heavy chain QVQLKE (SEQ ID No: 65) 2EVQLQQ (SEQ ID No: 66) 1 QVQLNQ (SEQ ID No: 67) 1 QVQLTQ (SEQ ID No: 68)1

The protein sequences of the variable light chain VL and variable heavychain VH are shown in FIGS. 1A and 1B, respectively. The hypervariablecomplementarity-determining regions (CDRs) are underlined. Thecorresponding DNA sequences are shown in FIGS. 2A and 2B, respectively.

Example 2 Generation of a Chimeric B-N10 Antibody

The identified variable sequences of the heavy and light antibody chainfrom Example 1 were cloned into a vector system for the expression ofrecombinant antibodies. The first step was to clone the sequences into aBS leader; the N terminal adds a secretion signal and the C terminaladds a splice-donor sequence. The second step was to clone thesesequences into the expression vectors which contain the constant humankappa chain and the constant human gamma-1 chain respectively. Thevector for the light chain and the vector for the heavy chain wereprepared and then transiently co-transfected into COS-7 cells by calciumphosphate precipitation or by lipofection. The cell culture supernatantwas harvested after 2 days. After expression of the chimeric B-N10 inCOS-7 cells and detection of an antibody titre in the supernatant(sandwich ELISA), its specific binding capacity to human interleukin 10(R&D Systems, Cat. No. 217-IL/CF, Lot ET114021, stored at −20° C.) wastested in the ELISA.

For the sandwich ELISA a mouse anti-human kappa chain antibody (BectonDickinson) was bound to the plate surface as a catcher antibody, thenincubated with cell culture supernatant and the presence of the chimericantibody was detected with a POD-conjugated rabbit anti-human IgG (H+L)antibody (Dianova). A chimeric control antibody in definedconcentrations (0.125 to 12 μg/mL) was used as a positive control.

For the antigen ELISA, human IL-10 was bound to the plate surface in aconcentration of 0.5 and 5 μg per mL. After incubation with the cellculture supernatant (undiluted and diluted 1:5), the binding of thechimeric B-N10 was detected with the POD-conjugated rabbit anti-humanIgG (H+L) antibody (Dianova). The murine B-N10 was used as a positivecontrol. The antibody was used in a concentration of 0.5 and 5 μg per mLand binding was detected with a POD-conjugated rabbit anti-mouse IgG/IgMantibody (Dako).

The results of the ELISA are discussed in Example 3.

Example 3 Humanization of Anti-IL 10 Antibodies

Initial efforts to reduce the immunogenicity of rodent antibodies inhumans involved the generation of chimeric antibodies by replacingrodent by human constant antibody domains. As the rodent frameworkregions within the variable domains might still induce an immuneresponse the more advanced method of CDR grafting was developed, meaningthe transfer of the antigen binding sequences (complementaritydetermining regions, CDR) onto completely human antibody frameworks(humanization). Usually human acceptor frameworks are selected thatresemble most closely the murine donor antibody to increase theprobability of restoring the original antigen specificity and affinityduring the humanization process. Different approaches using humanantibody germline sequences, consensus sequences of expressedantibodies, analysis of CDR loop structures and X-ray structures ofantibody/antigen complexes might be used or combined to improve theprocess. Usually several humanized antibody variants are generated inthis way and analysed afterwards regarding their biological effectswhich might differ from each other and the original antibody. Finallyaccording to the desired function of the antibody a suitable humanconstant region might be selected.

3.1 Sequence Comparisons between the Murine Variable Sequences of B-N10and Human Sequences, and Design of a Set of Humanized VL (hVL) and VH(hVH) Sequences

The murine anti-IL-10 antibody B-N10 was selected (Llorente et al., Eur.Cytokine Netw. 1993 November-December; 4(6): 421-7; and Llorente et al.,Arthritis Rheum. 2000 August; 43(8): 1790-80). The method for obtaininghumanized antibodies is based on the CDR-grafting procedure where thecomplementary determining regions (CDRs) are fused to human acceptorregions.

The choice of human acceptor frameworks was based on a combined analysisof three data sets:

1. The homology of the murine sequences to human germline sequences tominimize risk of somatic mutations:

2. The comparison of the murine sequences to human consensus sequencesto identify unusual amino acid residues and

3. The identification of the canonical structure classes of the CDRsequences to obtain information about important structural frameworkamino acid residues.

The murine variable light chain of the B-N10 shows the highest homologyto the human germline variable segment 2-30*01 (A17 (SEQ ID No: 14)) andto the joining segment JK1 (SEQ ID No: 15). The human consensus sequencewith highest homology to B-N10 is HuKII. Complementarity determiningregions (CDRs) of the variable light chain could be classified in caseof L1 to class 4, and in cases of L2 and L3 to class 1. Critical aminoacid residues were identified.

Sequence comparison between mouse CDR and human germline VL genes withthe canonical structure of class 4-1-1 revealed highest homology with2-30*01 (the lowest number of mismatching amino acids).

The murine variable heavy chain of the B-N10 shows the highest homologyto the human germline variable segment VH3-33 and to the joining segmentJH4 (SEQ ID No: 17). The human consensus sequence with highest homologyto B-N10 is HuHIII. Complementarity determining regions (CDRs) of thevariable heavy chain could be classified in case of H1 and H2 toclass 1. Critical amino acid residues were identified. Sequencecomparison between mouse CDR and human germline VH genes with thecanonical structure of class 1-1 revealed highest homology with 3-66*04(SEQ ID No: 16) (the lowest number of mismatching amino acids).Therefore, the germline sequence VH3-66 was taken too in consideration.

All data obtained were considered to design a set of different variablesequences of humanized variable light (12 variants) and variable heavychains (29 variants).

3.2 Construction of a Small Library and Selection of Humanized hIL-10Binding Antibody Fragments

In order to generate a library of potentially hIL-10 binding antibodyfragments to achieve the optimal human IL-10 binding antibody, the cDNAsequences coding for the 12 hVL and the 29 hVH fragments, as shown inFIG. 3, were generated under consideration of the codon usage ofeucaryotic cells.

The obtained cDNAs were cloned subsequently into cloning vectors andsequenced at SEQ Laboratories (Gottingen, Germany). The library wasconstructed in a way, that each of the 12 cDNAs coding for the hVLfragments were combined with the 29 cDNAs coding for the hVH fragmentsresulting in 348 potentially expressed antibody fragments.

Following the bacterial expression and two rounds of selection on humanIL-10 (R&D Systems, Cat.-No 217-IL/CF) the antibody fragments wereanalysed by ELISA for binding to hIL-10 (same as for selection). Inbrief, Maxisorb plates (Nunc, Germany) were coated with 1 μg/ml hIL-10in PBS over night at 4° C. After blocking and washing of the plates, thesupernatants of the antibody fragment producing bacteria were added. Fordetection of bound humanized antibody fragments a POD conjugatedsecondary antibody was used.

The coding sequences of good binders were analyzed and the occurrence ofidentified hVL- and hVH-fragments listed (Table 2).

TABLE 2 Occurrence of VL and VH fragments present in antibody fragmentsbinding to hIL-10. Occurrence of Occurrence of variable heavy variablelight chain variants chain variants hVH variant Occurrence hVL variantOccurrence hVH1 1 hVL1 2 hVH5 1 hVL2 1 hVH7 2 hVL3 1 hVH9 2 hVL5 1 hVH121 hVL6 3 hVH13 2 hVL7 4 hVH14 1 hVL8 18 (hVH16) 4 (hVL9) 4 hVH18 4 hVL101 hVH20 4 hVL11 1 hVH21 1 hVL12 2 hVH23 1 hVH26 9 hVH27 2 hVH28 3 hVH291

Sequences marked in bold were selected for subcloning into theappropriate eukaryotic expression vectors to analyze the bindingproperties in the context of the entire antibody. The sequences shown inbrackets were selected for subcloning into the expression vectors butthe procedure only resulted in defective constructs.

3.3 Generation of Expression Vectors for the Selected Humanized Lightand Heavy Chain Variants of BT-063

Based on the statistics determined by the screening approach a set ofhumanized VL and humanized VH variants of BT-063 were selected forcloning into a vector system. In a first step, the cDNAs encoding thehumanized VL and VH variants were transferred into an appropriate vectorin order to fuse a sequence coding for a secretory signal 5′ and asplice donor sequence 3′ to the cloned cDNA. These cDNA constructs were,in a second and final subcloning step, transferred into the expressionvectors encoding the human constant kappa and the human constant gamma-1chain, respectively. Plasmids of independently obtained hVL and hVHcontaining expression vectors were prepared by the endotoxin-free QiagenMidi-prep kit (Qiagen, Germany).

3.4 Transient Expression of the Selected Humanized BT-063 Variants inCOS-7 Cells and Comparison of Antibody Binding Towards hIL-10

For the transient expression of the humanized antibody variants in COS-7cells each of the selected humanized VL variants (hVL7 and hVL8) wascombined with each of the selected humanized VH variants (hVH1, hVH9,hVH13, hVH18, hVH20, hVH26, hVH28) resulting in 14 different humanizedantibodies.

In brief, the expression vectors coding for the light chain and for theheavy chain were transiently cotransfected into COS-7 cells by calciumphosphate precipitation in DMEM containing 10% FCS in a 24-well format.After transfection the medium was replaced by the serum free mediumCHO-S-SFM II (Invitrogen, Germany) and the supernatants of the COS-7cells were collected 2-3 days after transfection. The antibody titer ofthe humanized antibodies secreted into the supernatants of transfectedCOS-7 cells were analyzed by a sandwich ELISA. Based on the determinedantibody concentrations supernatants of all samples were adjusted to thesame antibody concentrations, and all samples were used to analyzebinding to human IL-10 in an antigen ELISA, whereby Maxisorb plates(Nunc, Germany) were coated with 2 μg/ml hIL-10 in PBS.

As shown in FIG. 4, all analyzed variants bind to hIL-10, however withdifferent binding properties. Significantly the highest signals in theantigen ELISA were obtained with the BT-063 variants hVH20/hVL7,hVH26/hVL7 and hVH20/hVL8 showing signal intensities comparable to thatobtained with the chimeric B-N10 antibody. Within these three antibodiesvariations in signal intensities (higher signal for hVH20/hVL8 and lowersignals for hVH20/hVL7 and hVH26/hVL7) could be caused by divergentantibody concentrations as a result of the quantifying sandwich ELISA(see above). All other investigated variants resulted in rather weaksignals compared to the chimeric B-N10 antibody.

3.5 Production and Affinity Purification of the Chimeric and HumanizedAntibody Variants

The selected humanized BT-063 variants (hVH20/hVL7, hVH20/hVL8,hVH26/hVL7) and the chimeric cB-N10 (discussed in Example 2) wereproduced in COS-7 cells.

Transient expression was performed as described in section 3.4 whereby10 cm tissue plates were used. Serum-free supernatants of approximately0.5 L of each variant were collected 5 days post transfection.

Purification of the antibodies was performed by protein A affinitychromatography from serum free supernatants. Supernatants were loaded inthe presence of 2M NaCl. Antibodies were eluted by a 0.1M Citrat bufferpH 4.0 and fractionated into tubes containing 2M phosphate buffer pH7.2. Buffer exchange against PBS as well as concentration of individualantibody probes was performed by centrifugation using membranes of a 30kDa cut off. The quality of purified materials was checked by antigenELISA, SDS-PAGE under non-reducing as well as reducing conditions and UVmeasurement at 260 nm and 280 nm.

Binding towards hIL-10 of the purified chimeric B-N10 and the humanizedvariants was tested by ELISA according to the method as described abovein Example 2. hIL10 was coated and the antibody binding was measured forthe variants cB-N10, BT-063-1 (hVH20/hVL7), BT-063-2 (hVH20/hVL8) andBT-063-3 (hVH26/hVL7). The results are shown in FIG. 5.

The signal intensities were comparable for the chimeric B-N10 and thehVH20/hVL7 variant, whereas the signal intensities of the variantshVH20/hVL8 and hVH26/hVL7 were slightly less.

3.6 Affinity Determination by Biacore Human IL-10

Surface plasmon resonance analysis was used to measure the associationand dissociation rate constants for binding of the different antibodies(murine, chimeric, 3 humanized variants) towards hIL-10 using BIACORE2000 (Biacore AB, Uppsala, Sweden). hIL-10 was immobilized on a CM-5sensor chip according to manufacturers conditions. hIL-10 wasimmobilized by adding a 50 μl aliquot of 20 μg/ml at a flow rate of 5μl/minute resulting in an immobilization density of 320 RU. Theimmobilized hIL-10 surface was regenerated in a two step cycle by using0.1M carbonate buffer pH 9.2 and 0.01M HCL/1M NaCl at flow rates of 50μl/minute for one minute each. Each antibody sample was analyzed atleast 4 times in antibody concentration ranges of 20-0.15 μg/ml.Calculations from the sensograms were performed by using the BIAevaluation version 3 (1999) software.

Table 3 summarizes the results of all Biacore measurements. All variantsbind comparable to hIL-10. However, slight differences are detectable.As a result the mouse monoclonal antibody B-N10, the chimeric cB-N10 aswell as the humanized variant BT-063-1 (hVH20/hVL7) bind with comparableaffinities whereas the two other humanized variants BT-063-2(hVH20/hVL8) and BT-063-3 (hVH26/hVL7) show reduced affinities (aboutfactor 3 compared to the murine B-N10). Slight differences inassociation and dissociation rates are also detectable.

TABLE 3 Results of Biacore measurements Antibody variant n ka [1/Ms] kd[1/s] KD [M] ± SD B-N10 6 4.43E6 2.05E−3 1.07E−9 ± 3.11E−10 cB-N10 46.23E5 8.48E−4 1.37E−9 ± 2.42E−10 BT-063-1 6 1.21E6 1.03E−3 1.22E−9 ±1.44E−10 BT-063-2 4 1.21E6 1.64E−3 2.81E−9 ± 1.03E−9  BT-063-3 5 1.07E62.66E−3 2.91E−9 ± 8.07E−10 n = number of individual measurements; ka =association rate; kd = dissociation rate; KD = dissociation constantCynomolgus IL-10

The affinity of the BT-63 variant 3 (hVH26/hVL7) to Cynomolgus IL-10 wasanalysed by additional surface plasmon resonance experiments using aBiacore T100 (Biacore AB, Uppsala, Sweden).

BT-063 was diluted in 10 mM acetate pH5.5 to 5 μg/mL and immobilizedusing amine coupling procedure to obtain a final level of about 1000 RU.Regeneration of the sensor chip surface was obtained injecting 10 mMGlycine-HCl pH 1.8 for 30 s. Samples were injected in differentconcentrations over the flow cell as well as over the reference cell.Signals obtained by the reference cell were subtracted from the signalsobtained by the detector flow cell and resulting binding profiles wereevaluated using a 1:1 Langmuir-binding model. A concentration dependingbinding profile was obtained and an average KD of 194 pM was calculatedfor Cynomolgus IL-10. As a positive control rhIL-10 was analysedresulting in a KD of 4.6 nM. Results are summarized in Table 4.

TABLE 4 Results of Biacore measurements with BT-063 Analyte Assay no ka[1/Ms] kd [1/s] KD [M] rhIL-10 1 6.0E5 0.3E−2  4.6E−9 rCIL-10 1 6.2E71.2E−2 0.196E−9 rCIL-10 2 8.6E7 1.7E−2 0.195E−9 rCIL-10 3 9.7E7 1.8E−20.191E−9 rhIL-10: recombinant human IL-10; rCIL-10: recombinantCynomolgus IL-10; ka = association rate; kd = dissociation rate; KD =dissociation constant

Example 4 Activity of Anti-IL 10 Antibodies In Vitro

To confirm the potency of BT-063, the blockade of IL-6 release inperipheral blood mononuclear cells (PBMCs) was examined. PBMCs releaseInterleukin-6 (IL-6) upon stimulation with Lipopolysaccharide (LPS). Aphysiological activity of Interleukin-10 (IL-10) is the inhibition ofsecretion of cytokines, e.g. IL-6. Thus, IL-10 addition to LPSstimulated cells inhibits IL-6 secretion, leading to a significantreduction of IL-6 present in the medium of the cell culture. However, asa consequence of BT-063 addition to the cell culture, IL-10 is bound andthus not able to bind to the receptor on the cell surface. Theinhibitory effect of IL-10 is compensated and IL-6 secretion isrestored, leading to IL-6 in the medium.

PBMCs were isolated from human blood by Ficoll gradient. The isolatedcells were seeded at 1×10⁶ cells/ml and stimulated with LPS for IL-6secretion, which was inhibited by addition of IL-10. The inhibitoryeffect of IL-10 was neutralized by addition of BT-063, thusreconstituting IL-6 secretion. Depending on the purpose (reference orlow, high quality control samples) of added BT-063, different titrationconcentrations of BT-063 were used, leading to concentration dependantsecretion of IL-6 which were detected in the supernatant of the cellculture.

TABLE 5 mean values of IL-6 levels from double determinations and IL-6reconstitution respectively in dependence of titration of referencestandard S1: 40 μg/mL concentration of BT-063 mean value of IL-.6Reconstitution of IL-6 [μg/mL] level [pg/mL] Secretion [%] 40.000 4254672.8% 20.000 43134 73.8% 13.333 37910 64.9% 8.889 31107 53.2% 5.92625602 43.8% 3.951 20793 35.6% 1.975 14200 24.3% 0.988 10227 17.5%

As shown in Table 5, the amount of secreted IL-6 is directly correlatedwith the concentration of BT-063. The higher the concentration ofBT-063, the more IL-6 was secreted from the PBMCs and thus present inthe supernatant. Incubation of the cells with 40 μg/mL BT-063 led to areconstitution of IL-6 secretion of about 73%, whereas with 0.988 μg/mLBT-063 (last step of titration) only 17.5% of the IL-6 level wasdetectable in the medium when compared to the positive control(stimulated PBMCs without IL-10 incubation).

Example 5 Different Effects of B-N10 and BT-063 on CytokineSynthesis/Level in Human Whole Blood Cultures

The immunopharmacological profile of BT-063 (variant hVH26/hVL7) wasevaluated by the drug-dependent modulation of experimentally inducedcytokine synthesis in human whole-blood cultures. With this method,direct and indirect influences of BT-063 on immune cell activities canbe determined. BT-063 activities were compared to the effects of B-N10.

The assays utilized whole-blood cultures from both healthy volunteersand SLE patients, which were analysed for cytokine release in thepresence of BT-063 or B-N10. Cytokine release by immune cells in wholeblood culture was examined in a resting state after incubation with theantibodies B-N10 or BT-063, respectively. Leukocytes from healthyvolunteers as well as cells from patients suffering from systemic Lupuserythematosus (SLE) have been included.

The cells for the experiments described in the following were obtainedby simple bleeding of the volunteers or the SLE patients and theirincubation with BT-063 or B-N10 respectively was done for 2 days inmicroculture plates.

The mediators measured in these experiments were mainly cytokines andchemokines, associated for example with Th-1-, Th-2- ormonocyte/macrophage-activation, such as Interferon gamma (IFN-gamma),interleukin-1 beta (IL-1beta), IL-12, IL-4, IL-8, or tumor necrosisfactor alpha (TNF-alpha). The concentration of each parameter to betested in the culture supernatants were determined using amultiparametric bead-based readout system (Luminex™-based technology,called multi-analyte profile, or MAP, tests Rules-Based Medicine, RBM(Austin, Tex., USA)). These assays were performed by EDI GmbH,Reutlingen, Germany The Luminex™ technology works in a manner similar toa mixture between ELISA and flow cytometry and counts 100 beads peranalyte, so the individual concentrations are back-calculated from themean fluorescence intensity of 100 individual measurements.

Only at antibody concentrations of 50 μg/ml, an induction of cytokinerelease has been observed.

Absolute cytokine levels measured in cell cultures from healthy donorsand from SLE patients incubated with B-N10 or BT-063 are summarized inTables 6 and 7 below.

TABLE 6 Summary of the cytokines which are differentially regulated byB-N10 or BT-063 in whole blood cultures of healthy donors. Absolutevalues of cytokines +/− SD (in ng/ml for IL1 alpha, MMP-2, in pg/ml forall other cytokines) released after incubation with 50 μg/ml antibodyconcentrations. Healthy volunteers induced in unstimulated cultures at50 μg/ml BT063 B-N10 Mean +/−SD Mean +/−SD T cell cytokines IFN gamma 2415 1757 1545 IL-2 0 0 19 12 IL-17 20 4 116 51 IL-4 22 4 96 4 T cellsurvival IL-7 103 15 319 31 Chemokines Mip1-beta 5453 852 316667 134656Mip1-alpha 154 34 6227 5763 MCP-1 1852 1001 120933 30897 IL-8 2093 26665967 26515 Proinflam- matory cytokines IL-6 20 2 20400 11557 IL1-alpha0.133 0.008 0.356 0.142 IL1 beta 44 16 3160 2936 TNF alpha 24 9 1068 386Anti- Inflam- matory IL-10 3 1 146 62

TABLE 7 Summary of differentially regulated cytokines (absolute values)by B-N10 or BT-063 in whole blood cultures of SLE patients. Absolutevalues of cytokines (in ng/ml for IL1 alpha, MMP-2, in pg/ml for allother cytokines) released after incubation with 50 μg/ml antibodyconcentrations. BT063 B-N10 Mean +/− Mean +/− T cell cytokines IFN- 1 1323 407 gamma IL-2 0 0 17 5 IL-4 28 1 118 80 IL-17 16 13 75 91 T cellsurvival IL-7 75 11 290 166 Chemokines IL-8 8410 9744 121100 145523 Mip1100 9 10235 12961 alpha Mip1 beta 3695 1407 156350 82944 MCP-1 2580 2560600500 663973 Proinflammatory cytokines IL-6 227 284 23200 17253IL1-beta 69 27 1420 438 Anti-inflammatory IL-10 8410 9744 121100 145523IL-1 alpha 0.072 0.048 0.206 0.013 TNF alpha 7 0 572 200 TNF beta 0 0 6642 Matrix metallo proteinases MMP-2 27 12 365 203

FIGS. 6A and B, 7A and B, 8A and B, and 9A and B show the differentialregulation of proimflammatory cyokines in human whole blood culturesfrom both healthy volunteers and SLE patients under non-stimulatingconditions. The figures show that whole blood cultures from healthyvolunteers and SLE patients display higher TNFalpha, IL-6, IL-1beta andIFN-gamma release after incubation with B-N10 compared to BT-063 (at 50μg/ml). It is noted that besides the proinflammatory function IL-6 actsin addition as factor for B-cell differentiation into plasma cells.

Remarkably it can be seen from these results that IL-10 andpro-inflammatory cytokines are upregulated to a higher extent by B-N10compared to BT-063 incubated cultures from both healthy and SLE donors,suggesting a better safety profile. This would be not expected as aresult upon humanization procedure.

Example 6 X-Ray Crystallography

6.1 Crystallisation of BT-063 Fab in Complex with Human IL-10

Several constructs of IL-10 were designed according to publishedstructural data (Zdanov et al., Structure, Vol. 3, 1995, pp. 591) andcloned by standard procedures into vectors for heterologous expressionin E. coli. Test expressions of the cloned constructs were performedaccording to standard protocols and showed a high over-expression forIL-10 as indicated by an increase in a band in the expected range ofaround 18 kDa.

IL-10 protein expressed under optimised conditions yielded viableamounts for subsequent protein purification. After refolding, theprotein was purified by immobilised affinity chromatography, sizeexclusion chromatography and ion exchange chromatography to yieldprotein with over 95% homogeneity as judged by Coomassie-stainedSDS-PAGE. The yield of purified protein was approximately 0.3 mg perliter expression culture, which was sufficient for crystallisationtrials.

The Fab fragment of BT-063 (variant hVH26/hVL7) was cleaved from theintact antibody using the protease papain and purified by protein A.Subsequently the Fab fragment was further purified by size exclusionchromatography.

The IL-10:BT-063 Fab complex was formed by mixing the purified proteins,with a molar excess of IL-10 and further purification by size exclusionchromatography. The retention volume was consistent with the size of thecomplex.

The protein was subsequently concentrated to concentrations suitable forcrystallisation.

Crystals of the IL-10:BT-063 Fab complex were prepared by the method ofco-crystallisation.

6.2 Data Collection and Processing

Crystals were flash-frozen and measured at a temperature of 100 K. TheX-ray diffraction data have been collected from co-crystals of IL-10with the Fab fragment of BT-063 at the SWISS LIGHT SOURCE (SLS,Villigen, Switzerland) using cryogenic conditions.

The crystals belong to space group P6 with two complexes in theasymmetric unit. Data were processed using the programmes XDS andXSCALE. Data collection statistics are summarised in Table 8.

TABLE 8 Statistics of data collection and processing ComplexIL-10:BT-063 X-ray source PX (SLS¹) Wavelength [Å] 1.007 DetectorPILATUS Temperature [K] 100 Space group P 6 Cell: a; b; c [Å] 219.00;219.00; 64.36 α; β; γ [°] 90.0; 90.0; 120.0 Resolution [Å]² 3.48(3.72-3.48) Unique reflections² 21124 (3817) Multiplicity² 3.0 (2.9)Completeness [%]² 91.2 (92.3) R_(sym)[%]^(2,3) 10.5 (44.0)R_(meas)[%]^(2,4) 14.8 (62.2) I/σ I² 6.1 (1.7) mean(I)/sigma^(2,5) 7.0(1.7) ¹SWISS LIGHT SOURCE (SLS, Villigen, Switzerland) ²Numbers inbrackets correspond to the highest resolution bin.$\;^{3}R_{sym} = {{\frac{\left. {\sum\limits_{h}\;\sum\limits_{l}^{n_{h}}}\; \middle| {{\hat{I}}_{h} - I_{h,i}} \right|}{\sum\limits_{h}\;{\sum\limits_{l}^{n_{h}}\; I_{h,i}}}\mspace{14mu}{with}\mspace{14mu}{\hat{I}}_{h}} = {\frac{1}{n_{h}}{\sum\limits_{l}^{n_{h}}\; I_{h,i}}}}$where I_(h,i) is the intensity value of the ith measurement of h$\;^{4}R_{meas} = {{\frac{\left. {\sum\limits_{h}\;{\sqrt{\frac{n_{h}}{n_{h} - 1}}\sum\limits_{l}^{n_{h}}}}\; \middle| {{\hat{I}}_{h} - I_{h,i}} \right|}{\sum\limits_{h}\;{\sum\limits_{l}^{n_{h}}\; I_{h,i}}}\mspace{14mu}{with}\mspace{14mu}{\hat{I}}_{h}} = {\frac{1}{n_{h}}{\sum\limits_{l}^{n_{h}}\; I_{h,i}}}}$where I_(h,i) is the intensity value of the ith measurement of h⁵Calculated from independent reflections6.3 Structure Modelling and Refinement

The phase information necessary to determine and analyse the structurewas obtained by molecular replacement. Published models of IL-10 and aFab fragment were used as a search model. Subsequent model building andrefinement was performed according to standard protocols with thesoftware packages CCP4 and COOT. For the calculation of the freeR-factor, a measure to cross-validate the correctness of the finalmodel, 4.2% of measured reflections were excluded from the refinementprocedure (see Table 9).

The nanobody parameterisation was carried out with the programmeCHEMSKETCH. LIBCHECK (CCP4) was used for generation of the correspondinglibrary files.

The water model was built with the “Find waters . . . ”-algorithm ofCOOT by putting water molecules in peaks of the Fo-Fc map contoured at3.0σ followed by refinement with REFMAC5 and checking all waters withthe validation tool of COOT. The criteria for the list of suspiciouswaters were: B-factor greater 80, 2Fo-Fc map less than 1.2σ, distance toclosest contact less than 2.3 Å or more than 3.5 Å. The suspicious watermolecules and those in the active site (distance to inhibitor less than10 Å) were checked manually. The occupancy of side chains, which were innegative peaks in the Fo-Fc map (contoured at −3.0σ), were set to zeroand subsequently to 0.5 if a positive peak occurred after the nextrefinement cycle.

The Ramachandran Plot of the final model shows 80.8% of all residues inthe most favoured region, 17.9% in the additionally allowed region, 0.7%of the residues in the generously allowed. Residues Val86(A), His14(B),Asp86(B), Ser131(C), Val56(D) and Val56(F) are found in the disallowedregion of the Ramachandran plot (Table 9). They are either confirmed bythe electron density map or could not be modelled in another sensibleconformation. Statistics of the final structure and the refinementprocess are listed in Table 9.

TABLE 9 Refinement statistics ¹ Complex IL-10:BT-063 Resolution [Å]20.0-3.48 Number of reflections (working/test) 20199/889 R_(cryst) [%]29.7 R_(free) ² [%] 35.5 Total number of atoms: Protein 8870 WaterLigand — Deviation from ideal geometry: ³ Bond lengths [Å] 0.007 Bondangles [°] 0.93 Bonded B's ⁴ [Å²] 0.0 Ramachandran Plot: ⁵ Most favouredregions 80.8 Additional allowed regions 17.9 Generously allowed regions0.7 Disallowed regions 0.6 ¹ Values as defined in REFMAC5, without sigmacut-off ² Test-set contains 4.2% of measured reflections ³ Root meansquare deviations from geometric target values ⁴ Calculated withprogramme MOLEMAN ⁵ Calculated with programme PROCHECK6.4 X-Ray Structure Analysis

The complex structure of human IL-10 bound by BT-063 Fab antibodyfragment was analysed at a resolution of 3.48 Å and reveals the detailedbinding mode of the Fab antibody fragment.

The resulting electron density shows an unambiguous binding mode for theFab fragment, including the orientation and conformation of the Fabfragment. The crystal of space group P6 contains two complexes in theasymmetric unit.

The structure of IL-10 in complex with Fab is represented in FIG. 6. TwoFab fragments bind with their CDR loops to each homodimer of IL-10.

The following residues of IL-10 (molecules A and B) can be found in thevicinity of the CDR loops within a maximum distance of 3.9 Å: Arg27, Lys34, Gln38, Met39, Asp41, Gln42, Asp44, Leu46, Glu50, Leu53, Glu142,Asp144, Ile145, Asn148, Tyr149, Glu151, and Thr155.

The following residues of the CDR loops can be found in the vicinity ofthe IL-10 within a maximum distance of 3.9 Å: Phe27, Ser28, Ala30,Thr31, Tyr32, Trp52, Arg53, Gly54, Ser56, Asn73, Ser74, Tyr100, Gly101,Tyr103 (molecules C and E), Ser32, Asn33, Asn35, Tyr37, Lys55 (moleculesD and F).

The binding site of BT-063 coincides with the binding site of the IL-10receptor on the surface of IL-10 as shown by overlaying the complexstructure of IL-10:BT-063 with a published structure of theIL-10:IL-10R1 receptor complex (FIG. 7).

The BT-063 amino acid residues in contact with human IL-10 as identifiedby X-ray analysis are highlighted on the linear amino acid sequence ofBT-063 variable antibody domains shown below.

BT-063 VL (SEQ ID No: 69) DVVMTQSPLS LPVTLGQPAS ISCRSSQNIV HSNGNTY LEWYLQRPGQSPR LLIY KVSNRF SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCFQGSHVP WTFGQGTKVE IK BT-063 VH: (SEQ ID No: 70)EVQLVESGGG LVQPGGSLRL SCAASGFSFA TYGVHWVRQS PGKGLEWLGV IWRGGSTDYS AAFMSRLTIS KDNSKNTVYL QMNSLRAEDT AVYFCAKQAY  GHYMDYWGQG TSVTVSS

CDR regions (Honegger and Plückthun (2001) J. Mol. Biol., 309, 657-670)are underlined (CDR1, CDR2 and CDR3 of the light chain are SEQ ID Nos:71, 72 and 73, respectively; CDR1, CDR2 and CDR3 of the heavy chain areSEQ ID Nos: 74, 75 and 76, respectively). Contact residues with IL-10are shown in bold.

Within the light chain contact residues are found in CDR1 and CDR2 butnot in CDR3. Regarding the heavy chain resides of all three CDRs areinvolved in antigen binding. Two residues of FR3 (Asn73 and Ser74) alsocontribute to antigen binding.

Ser28 and Ala30 at the beginning of CDR1 are part of the murine VHpredecessor sequence (BN-10) and not present in the selected humanframework (3-66*04). Both positions are less frequently involved inantigen binding and were introduced as alternative amino acids duringthe humanisation process.

Residues Asn73 and Ser74 are found in murine and frequently in humanantibody framework sequences but are usually not involved in antigenbinding. (www.bioc.uzh.ch/antibody; Honegger and Plückthun, 2001). Theircontribution to antigen binding is unexpected.

IL-10 amino acid residues involved in BT-063 binding are shown below.Also indicated are residues of IL-10 involved in binding to the highaffinity IL-10 receptor chain (IL-10R1) and the low affinity receptorchain (IL-10R2). Both receptor chains are involved in the binding of aIL-10 homodimer and necessary for signaling. In sequence A the residueswhich contact BT-063 are shown in bold. In sequence B the contactresidues to IL-10R1 are marked in bold, contact residues to IL-10R2 aremarked in italics and underlined and contact residues shared by IL-10R1and 2 are marked in as being in bold, italics and underlined (Pletnev etal 2005).

A (SEQ ID NO: 1) SPGQGTQSEN SCTHFPGNLP NMLRDL R DAF SRV K TFF QM K DQ LD N L LLK E  SL L EDFKGYL GCQALSEMIQ FYLEEVMPQAENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK LQEKGIYKAM S E FDI FI NY I  E AYM T MKIRN B (SEQ ID NO: 1) SPGQGTQSEN SCTHF P G N L P  N ML R DL R D AF S R VKTFF Q MK D QLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA E NQDPDIK AH  V NSLGENLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK LQEKGIY K AM  S EF D IFINYI  EAYMTMK I RN

It can be seen that BT-063 binds to a discontinuous epitope of IL-10comprising residues of helix A (Arg27, Lys34, Gln38, Met39, Asp41) andthe N-terminal part of helix B (Glu50 and Leu53) including theconnecting loop sequence (Glu42, Asp44, Leu46) of one IL-10 monomer aswell as residues of the helix F′ (Glu142, Asp144, Ile145, Asn148,Tyr149, Glu151, Thr155) of the second IL-10 monomer.

Example 7 In Vivo Single Dose Toxicity Study in Cynomolgous Monkey

A single dose toxicity study including safety pharmacology parameterswas performed in Cynomolgus monkeys following a single intravenousinjection of BT-063 (variant hVH26/hVL7). Animals were distributed intofour groups (placebo, 1 mg/kg, 7 mg/kg and 50 mg/kg) with 4animals/sex/group. BT-063 was intravenously injected on day 1. Half ofthe animals were necropsied after 5 days, while the remaining animalswere sacrificed on day 28.

The low dose level of 1 mg/kg in this study corresponds to a humanequivalent dose of ˜300 μg/kg, resulting in a total human dose of 18 mg(60 kg body weight). A similar dose of the BT-063 predecessor antibodyB-N10 had been applied daily for 21 days in a small investigatorinitiated trial (Llorente, 2000). This amount of antibody has shownpharmacological effects and clinical efficacy. The low dose level wastherefore chosen accordingly. The high dose was chosen as a multiple (50μg) of the starting dose. The intermediate dose is the geometric mean ofthe low and the high doses.

During the study no toxicologically significant or relevant changes inphysiological or histopathology parameters were observed. In addition,changes in clinical chemistry parameters were considered of little or notoxicological significance.

Example 8 In Vivo Single Dose Toxicity Study in Healthy Volunteers

A study was conducted to monitor the safety and tolerability of BT-063(variant hVH26/hVL7), and the effects of BT-063 administration, usingescalating doses of the antibody in healthy volunteers. Twenty-threevolunteers received a single intravenous administration of BT-063, in 8dosage groups. The dosage groups were as follows: 0.175 mg, 0.75 mg, 3.5mg, 7 mg, 15 mg, 30 mg, 60 mg and 100 mg. There were three volunteersper group, except for the 100 mg dose group where there were twovolunteers.

Each dose was diluted with 0.9% sodium chloride injection up to a totalvolume of 20 ml. The dose is administered as a single continuousintravenous infusion over 2 hours.

The volunteers were assessed over a period of 85 days after theinjection and blood was taken at multiple time points over this period.

Cytokines

From the plasma taken, assessments were made on the levels of cytokinesIFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNFα before andafter infusion of BT-063.

Table 10 shows the upper limit of normal values for IL-6, IL-8 and IL-10calculated from predose and screening values.

TABLE 10 Cytokine concentration in healthy volunteers Cytokine (pg/mL)IL-6 IL-8 IL-10 Mean 5.6 12.4 4.9 SD 10.6 11.5 9.3 ULN 26.7 35.3 23.5(Mean + 2 × SD) Range (lowest to 0-44.5 0.0-45.1 0.0-40.2 highestvalues)

Further results from the cytokine measurements are shown in FIGS. 12 and13.

A non-dose dependent transient increase of IL-6 and IL-8 was presentwithin the 24 hours post infusion, returning to pre-dose levels after 3days. This effect is thought to be associated with the infusion eventrather than with the antibody since there was no dose relationship andthe effect occurred already at the lowest dose

Surprisingly, given that IL-10 is an important regulatory cytokine, noincrease in levels of IFNγ, IL-1β, IL-2, IL-4, IL-5, and TNFα weredetected as shown in FIG. 12. The lack of elevation in levels of IL-4,IL-5 IFN-γ, IL-2 also confirm that there is no T-helper cell activationresulting from the BT-063 administration. Further, since bodytemperature is also an indication of the large-scale release ofpro-inflammatory cytokines, this parameter was also measured in thetreated volunteers. However, no increase in body temperature wasdetected after administration.

As shown in FIG. 13, only IL-10 plasma concentration is influenced byadministration of BT-063, with detected plasma IL-10 increasing as thedose of BT-063 is increased. It is noted that the assay used detectsboth free IL-10 and IL-10 bound to BT-063. There are two possibleexplanations for the increase: (1) a prolonged half life of IL-10, withbinding of IL-10 to BT-063 preventing IL-10 internalization, andconcomitantly no elimination of IL-10 from the blood (there is normallya rapid turnover of IL-10, with the half life of secreted IL-10 beingapproximately 2.3-3.5 hours (Huhn et al., Blood (1996) January 15:87(2): 699-705)); and/or (2) induction of a negative feedback loop—withBT-063 blocking the binding of IL-10 to its receptor no cellular uptakeof IL-10 is possible, triggering B-cells to produce more IL-10. Weconsider that scenario (2) is more probable since it is confirmed by invitro data taken from whole blood culture experiments with BT-063 andmurine IL-10R knockout cells (Mahnke et al., unpublished data).

Pharmacokinetics

The pharmacokinetic data showed that the Cmax, AUC and half-life ofBT-063 are in the range of the expected theoretical values. The terminalhalf-life of BT-063 being between 15 and 30 days. After administrationof doses of 30 mg or higher, BT-063 is still detectable in the plasmaafter 85 days.

No human anti-human antibodies (HAHA) were observed in the treatedvolunteers, despite the fact that HAHA responses have been observed withother cytokine neutralizing antibodies (e.g. with Adalimumab, ahumanized anti-TNF alpha).

Despite the increase in detected amounts of IL-10 after administrationof BT-063 it is noted that this study demonstrates the safety andtolerability of even large dosages of BT-063 and thus it can beconcluded that sufficient dosages of BT-063 can be safely administered(in particular, the absence of an intolerable increase in the level ofpro-inflammatory cytokines) to SLE patients to counteract the effects ofexcess IL-10.

The invention claimed is:
 1. An in vitro method for neutralizinginterleukin-10 in a sample, comprising a step of contacting the samplewith an antibody or fragment thereof capable of binding interleukin-10,so as to bind the antibody or fragment thereof to the interleukin-10,wherein the antibody or fragment thereof comprises a variable lightchain comprising the amino acid sequences SEQ ID NO: 71, 72 and 73, anda variable heavy chain comprising the amino acid sequences SEQ ID NO:74, 75 and
 76. 2. An in vitro method for detecting the presence ofinterleukin-10 in a sample comprising a step of contacting an antibodyor fragment thereof with the sample, washing to remove antibody orantibody fragments which are not bound to the sample, and detecting thepresence of the antibody or fragment in the sample, wherein saidantibody or fragment thereof is capable of binding interleukin-10,wherein the antibody or fragment thereof comprises a variable lightchain comprising the amino acid sequences SEQ ID NO: 71, 72 and 73, anda variable heavy chain comprising the amino acid sequences SEQ ID NO:74, 75 and
 76. 3. An in vitro method for detecting the presence ofinterleukin-10 in a sample comprising a step of contacting a labeledantibody or fragment thereof with the sample, washing to remove antibodyor antibody fragments which are not bound to the sample, and detectingthe presence of the label in the sample, wherein said antibody orfragment thereof is capable of binding interleukin-10, and wherein theantibody or fragment thereof comprises a variable light chain comprisingthe amino acid sequences SEQ ID NO: 71, 72 and 73, and a variable heavychain comprising the amino acid sequences SEQ ID NO: 74, 75 and
 76. 4.The method of claim 1, wherein the antibody or fragment thereof iscapable of being administered to a subject without causing anintolerable increase in the level of pro-inflammatory cytokines.
 5. Themethod of claim 1, wherein the antibody or fragment thereof comprises avariable light chain having SEQ ID NO: 69 or a variable heavy chainhaving SEQ ID NO:
 70. 6. The method of claim 1, wherein the antibody orfragment thereof comprises a variable light chain having SEQ ID NO: 69and a variable heavy chain having SEQ ID NO:
 70. 7. The method of claim1, wherein the antibody further comprises a constant region of an IgGisotype.
 8. The method of claim 2, wherein the antibody or fragmentthereof is capable of being administered to a subject without causing anintolerable increase in the level of pro-inflammatory cytokines.
 9. Themethod of claim 2, wherein the antibody or fragment thereof comprises avariable light chain having SEQ ID NO: 69 or a variable heavy chainhaving SEQ ID NO:
 70. 10. The method of claim 2, wherein the antibody orfragment thereof comprises a variable light chain having SEQ ID NO: 69and a variable heavy chain having SEQ ID NO:
 70. 11. The method of claim2, wherein the antibody further comprises a constant region of an IgGisotype.
 12. The method of claim 3, wherein the antibody or fragmentthereof is capable of being administered to a subject without causing anintolerable increase in the level of pro-inflammatory cytokines.
 13. Themethod of claim 3, wherein the antibody or fragment thereof comprises avariable light chain having SEQ ID NO: 69 or a variable heavy chainhaving SEQ ID NO:
 70. 14. The method of claim 3, wherein the antibody orfragment thereof comprises a variable light chain having SEQ ID NO: 69and a variable having chain having SEQ ID NO:70.
 15. The method of claim3, wherein the antibody or fragment thereof further comprises a constantregion of an IgG isotype.
 16. The method of claim 1, wherein saidantibody or fragment thereof is humanized.
 17. The method of claim 2,wherein said antibody or fragment thereof is humanized.
 18. The methodof claim 3, wherein said antibody or fragment thereof is humanized. 19.The method of claim 1, wherein said antibody or fragment thereof ischimeric.
 20. The method of claim 2, wherein said antibody or fragmentthereof is chimeric.
 21. The method of claim 3, wherein said antibody orfragment thereof is chimeric.